Leprosy or Hansen's disease is a chronic infectious disease that primarily affects the skin, peripheral nerves, upper respiratory tract and eyes (Sasaki et al., “Mycobacterium leprae and Leprosy: A Compendium,” Microbiol. Immunol 45:729-36 (2001)). The pathogen is an acid-fast bacillus, Mycobacterium leprae (M. leprae), that was first identified by the Norwegian physician, Gerhard Hansen in 1873. The bacilli proliferate in macrophages infiltrating the skin and gain entry to the dermal nerves via the laminar surface of Schwann cells where they replicate. After entry, the Schwann cells proliferate and then die. Combined with the ensuing host inflammatory response to the mycobacteria, damage results in the peripheral nerves which leads to functional impairment, including desensitization to temperature, light touch and pain. It appears that attachment to Schwann cells causes demyelination and proliferation of large numbers of mycobacterium in the cells (Rambukkana et al., “Contact Dependent Demyelation by Mycobacterium Leprae in the Absence of Immune Cells,” Science 296:927-931 (2002), Oliveira et al., “Expression of Toll-like Receptor 2 on Human Schwann Cells: A Mechanism of Nerve Damage in Leprosy,” Infection and Immun. 71:1427-1433 (2003), Kim et al., “Detection of Gene Mutations Related with Drug Resistance in Mycobacterium leprae from Leprosy Patients Using Touch-Down (TD) PCR,” FEMS Immunology and Medical Microbiology 36:27-32 (2003)).
Leprosy currently remains endemic in some developing parts of the world (Ishii et al, “Survey of Newly Diagnosed Leprosy Patients in Native and Foreign Residents of Japan” Int. J. Lepr. 68:172-6 (2000)). The WHO in 1991 wanted to eliminate leprosy by 2000 (World Health Organization. “World Health Assembly-Resolution,” WHA44.9 (1991)) (less than 1 case per 10,000). By 2000, 597,232 cases were registered and 719,330 cases were newly detected (World Health Organization. “Leprosy-Global Situation,” Weekly Epidemiological Record 75:225-232 (2000)). There have been 690,830 newly detected patients in 2001 with 91% in the top six countries where the disease is most prevalent. The prevalence rate in these top six countries has been estimated at 3.9 per 10,000, with a very uneven distribution. By 2001, India accounted for 78% (439,782 cases, 4.3 per 10,000), Brazil-12% (77,676 cases, 4.5 per 10,000), Nepal-1.7% (10,657 cases, 4.4 per 10,000), Myanmar-1.5% (8,237 cases, 1.8 per 10,000), Mozambique-1% (6,775 cases, 3.4 per 10,000) and Angola-0.6% (4,115 cases, 3.1 per 10,000). The diagnosis of leprosy is mainly based on the clinical signs and the symptoms of the disease, plus the results of skin smears. Patients showing negative smears at all sites are grouped as paucibacillary leprosy (PB), while those showing positive smears at any site are multibacillary leprosy (MB). The clinical classification also uses the number of skin lesions and nerves involved as the basis for grouping leprosy patients into PB and MB leprosy and to determine the treatment regimen (Ridley et al., “Classification of Leprosy According to Immunity—A Five Group System,” Int. J. Lepr. 54:255-73 (1966)). Bleharski et al. (Bleharski et al., “Use of Genetic Profiling in Leprosy to Discriminate Clinical Forms of the Disease,” Science 301:1527-1530 (2003)) using genetic expression profiling were able to correlate gene expression with clinical forms of leprosy. They found significant expression of leukocyte immunoglobulin-like receptor (LIR) genes in lepromatous leprosy and increased expression of Toll-like receptor 2 and 1 (LTRs) in tuberculoid leprosy (Krutzik et al., “Activation and Regulation of Toll-Like Receptors 2 and 1 in Human Leprosy,” Nature Medicine 9:525-532 (2003), Krutzik et al., “The Role of Toll-Like Receptors in Combating Mycobacteria,” Seminars in Immunology 16:35-41 (2004))
There are several effective chemotherapeutic agents against M. leprae. Dapsone (diaphenylsulfone, DDS), rifampicin, clofazimine, ofloxacin and minocycline constitute the multidrug therapy (MDT) regimen. Other effective chemotherapeutic agents include levofloxacin, sparfloxacin and clarithromycin (Sugita et al, “A Case of Relapsed Leprosy Successfully Treated with Sparfloxacin,” Arch. Deunatol 32:1397-1398 (1996), Ishii et al., “Sparfloxacin in the Treatment of Leprosy Patients,” Int. J. Dermatol 36:619-62 (1997), WHO. “Model Prescribing Information-Drug Used in Leprosy,” WHO/DMP/DSI/98.1 (1998), WHO. “Chemotherapy of Leprosy for Control Programmes,” WHO Technical report series 675 (1982), WHO. “WHO Expert Committee on Leprosy, Sixth Report,” WHO Technical report series 768 (1988), WHO. “Chemotherapy of Leprosy, Report of a WHO Study Group,” WHO Technical report series 847 (1994), WHO. “A Guide to Eliminating Leprosy as a Public Health Problem,” WHO/LEP/95.1 (1995), WHO. “WHO Expert Committee on Leprosy, Seventh Report,” Technical report series 874 (1998)). It has been proven that monotherapy will result in the development of resistance to the drug. Trials have shown that complete clearing of lesions takes 1-2 years after treatment discontinuation. There is evidence that 3-6 months of administration of MDT clears all live organisms. Resistance of M. leprae to anti-leprosy drugs has been reported world-wide. Drug resistance is due to genetic changes in drugs targeting genes for rifampicin (rpoB or β-subunit of RNA polymerase), dapsone (folP or dihydropteroate synthase), and ofloxacin (gyrA or DNA gyrase) (Williams et al., “PCR-Based Diagnosis of Leprosy in the United States,” Clin. Micro. Newsletter 25:57-61 (2003), You et al., “Mutations in Genes Related to Drug Resistance in Mycobacterium Leprae Isolates from Leprosy Patients in Korea,” J. Medicine 50:6-11 (2005), Maeda et al., “Multidrug Resistant Mycobacterium Leprae from Patients with Leprosy,” Antimicrobial Agents and Chemotherapy 45:3636-3639 (2001)). Patients with the tuberculoid type are relatively resistant to the pathogen with localized lesions that express the type-1 cytokines characteristic of cell-mediated immunity. Lepromatous leprosy is relatively susceptible to the organism with systemically disseminated and type-2 cytokines characteristic of humoral responses (WHO. “Chemotherapy of Leprosy for Control Programmes,” WHO Technical Report Series 675 (1982), Kang et al., “Differential Production of Interleukin-10 and Interleukin-12 in Mononuclear Cells from Leprosy Patients with a Toll-Like Receptor 2 Mutation. Immunology,” 112:674-680, (2004)).
Examining the genetic diversity of M. leprae is only at its infancy. In comparison to the M. tuberculosis genome, the M. leprae genome is smaller; has less G/C content; less protein-coding genes; more gene density and similar average gene length (Cole et al., “Massive Decay in Leprosy Bacillus” Nature 409:1007-11 (2001), Kato-Maeda et al., “Comparing Genomes Within the Species Mycobacterium Tuberculosis,” Genome Research 11:547-554 (2001), Rambukkana, A. “M. Leprae Genome Sequence,” Trends in Microbology 98:157 (2001)). There has been little evidence for deletion events or insertions as the cause of the smaller size in the M. leprae genome. Genetic diversity has been found for short tandem repeat loci. These include the TIC repeat of 10-37 repeats between two pseudogenes, a 6 bp (GACATC) repeat in the rpoT gene (3 or 4 repeats in Asia) and two newly described TA and AT repeats (Shin et al., “Variable Numbers of TTC Repeats in Mycobacterium Leprae DNA from Leprosy Patients and Use in Strain Differentiation,” J. Clinical Microbiology 38:4535-4538 (2000), Chae et al., “Typing of Clinical Isolates of Mycobacterium Leprae and Their Distribution in Korea,” Leprosy Review 73:41-46 (2002), Young, D. “Prospects for Molecular Epidemiology of Leprosy,” Leprosy Review. 74:11-17 (1993), Young et al., “Leprosy, Tuberculosis, and the New Genetics,” J. Bacteriology. 175:1-6 (1993), Cole et al., “Repetitive Sequences in Mycobacterium Leprae and Their Impact on Genome Plasticity,” Leprosy Review 72:449-461 (2001), Matsuoka et al., “Mycobacterium Leprae Typing by Genomic Diversity and Global Distribution of Genotypes,” International J. Leprosy 68:121-128 (2000)). Groathouse et al. (Groathouse et al., “Multiple Polymorphic Loci for Molecular Typing of Strains of Mycobacterium Leprae,” J. Clin. Micro 42:1666-1672 (2004)) have identified nine other potential short tandem repeats (STRs). Conclusive identification of the presence of M. leprae in a sample can be obtained by PCR-restriction fragment length polymorphism analysis of the heat shock 65 gene (hsp65) by digestion with BstEII and/or HaeIII followed by Polyacrylamide Gel Electrophoresis (PAGE) (Rastogi et al., “Species Specific Identification of Mycobacterium Leprae by PCR-Restriction Fragment Length Polymorphism Analysis of the Hsp65 Gene,” J. Clinical Microbiology 37:2016-2019 (1999)). Molecular epidemiology will make it possible to study the global and geographical distributions of M. leprae, explore the relationship between genotypes-incidence rates, mode of transmission and the type of disease (tuberculoid versus lepromatous). Cole et al., “Massive Decay in Leprosy Bacillus”, Nature 409:1007-1011 (2001). However, to date no methods are available for performing molecular epidemiology of M. leprae due to their lack of highly polymorphic loci.
The present invention is directed to overcoming these and other deficiencies in the art.